Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Opposing Effects of CTLA4 Insufficiency on Regulatory versus Conventional T Cells in Autoimmunity Converge on Effector Memory in Target Tissue

doi: 10.4049/jimmunol.1400876

Figure Lengend Snippet: (A) Representative intracellular flow cytometry plots of IFNγ- and IL17-producing CD4+ T cells in the PLN (numbers represent percentages of gated populations). (B–C) Frequencies and total cell numbers of CD4+IL17+ (B) and CD4+IFNγ+ (C) subsets impacted by CTLA4RNAi on the B6.H2g7 background. Control mice were transgene-negative littermates (n=4–6 per group). (D) Littermate CTLA4RNAi/B6.H2g7 mice were treated with non-specific ratIgG2a control antibody or anti-IL7Rα antibody for 3 weeks. Mice were monitored for diabetes for 40 days. (E) Representative intracellular flow cytometry plots of the pancreas showing IL17- and IFNγ-production by CD4+ T cells after anti-IL7Rα treatment (numbers represent percentage of gated populations). (F–G) Frequencies and total cell numbers of CD4+IL17+(F) and CD4+IFNγ+ (G) after anti-IL7Rα treatment (n=4–6 mice per group). Each data-point represents one animal (Mean ± SEM). *p<0.05; **p<0.01; ***p<0.005; ns, not significant.

Article Snippet: For anti-IL7Rα antibody treatment, CTLA4RNAi/B6.H2 g7 mice were intraperitoneally administered with anti-IL7Rα (A7R34) antibody or Rat IgG1 isotype control antibody (BioXcell, West Lebanon, NH) at a dose of 25ug/g body weight beginning at 3–4 days of age, twice a week for 3 weeks.

Techniques: Flow Cytometry, Control

Journal: Nature communications

Article Title: Context-specific regulation of surface and soluble IL7R expression by an autoimmune risk allele.

doi: 10.1038/s41467-019-12393-1

Figure Lengend Snippet: Fig. 4 Monocyte surface IL7R is functional, activating multiple transcriptional pathways. a Violin plot demonstrating monocytes from PBMC cultures in the untreated state, after exposure to LPS and after exposure to LPS with recombinant IL-7. IL-7 leads to marked downregulation of IL7R on LPS stimulated monocytes, indicative sensitivity to exogenous cytokine (paired t-test). b Volcano plot of mRNA from RNAseq experiments of 8 paired monocyte samples either treated with LPS alone for 24 h or with LPS with additional IL-7 added for the last 2 h of culture. Treatment leads to widespread differential transcript expression with the most significant 20 transcripts labelled. c Example boxplots of genes differentially regulated by recombinant IL-7 in monocytes (linear model). d t-SNE plot of single-cell real-time quantitative PCR (RT-qPCR) data from 10 individuals (1218 cells) either untreated or in the presence LPS showing expression of membrane-bound IL7R (IL7Rmb), soluble IL7R (sIL7R), CLEC4E, TNF, CUX1, AKT1, STAB1, CD163, CSF1R, CCL5, DDX39A, DDX39B, MALAT1, LTB, IFNB1, CD14, FCGR3B, LYZ, and ACTB. e Density plots of single-cell RT-qPCR expression of sIL7R and mbIL7R normalized expression in untreated and LPS treated monocytes according to rs6897932 genotype. Kolmogorov Smirnov test. f Violin plots of single-cell RT-qPCR expression of MALAT1 in LPS stimulated monocytes either alone or in the presence of recombinant human IL-7 according to rs6897932 genotype, t-test

Article Snippet: Plates were blocked with 5% BSA for 1 h, washed, and cell supernatants added for 2 h. Bound sIL7R was detected with 12.5 ng/ml biotinylated goat anti-human CD127 polyclonal Ab (R&D Systems) for 1 h, followed by a 30 min incubation with streptavidin-HRP and a 15 min incubation with TMB peroxidase substrate (Thermo Fisher).

Techniques: Functional Assay, Recombinant, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Membrane

Journal: Nature communications

Article Title: Context-specific regulation of surface and soluble IL7R expression by an autoimmune risk allele.

doi: 10.1038/s41467-019-12393-1

Figure Lengend Snippet: Fig. 5 IL7R+ monocytes form a distinct subset detectable in synovial fluid. a Representative flow cytometry of IL7R staining of spondyloarthritis matched patient PBMC (left) and SFMC (middle isotype control, right) gated on CD3-CD14+ as per sequential gating strategy shown in Supplementary Fig. 1. b Results from 4 patients demonstrating comparative monocyte IL7R staining in matched PBMC and SFMC using flow cytometry. c t-SNE clustering of monocytes from single-cell RNA sequencing of spondyloarthritis patient SFMC showing 5 clusters (n = 3). d Single-cell monocyte expression of IL7R, LTB, CD14, CCL5, IL32, and TCF7 overlaid on t-SNE clustering in c. e Volcano plot of genes significantly upregulated in the SFMC monocyte cluster 4 compared to all other SFMC monocyte clusters with top 20 genes annotated. Wilcoxon rank sum test. f Heatmap of top 10 genes from the each of the five SFMC monocyte clusters identified in c

Article Snippet: Plates were blocked with 5% BSA for 1 h, washed, and cell supernatants added for 2 h. Bound sIL7R was detected with 12.5 ng/ml biotinylated goat anti-human CD127 polyclonal Ab (R&D Systems) for 1 h, followed by a 30 min incubation with streptavidin-HRP and a 15 min incubation with TMB peroxidase substrate (Thermo Fisher).

Techniques: Cytometry, Staining, Control, RNA Sequencing, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Dual roles of IL-15 in maintaining IL-7RalphalowCCR7- memory CD8+ T cells in humans via recovering the phosphatidylinositol 3-kinase/AKT pathway.

doi: 10.4049/jimmunol.179.10.6734

Figure Lengend Snippet: FIGURE 1. IL-15R and IL-2/15R expression by IL-7Rhigh and IL- 7RlowCCR7 memory CD8 T cells. PBMCs were stained with anti- CD8, CCR7, CD45RA, IL-7R, IL-15R, IL-2/15R, or isotype Abs and analyzed on a flow cytometer (A–E). A, CCR7 memory CD8 T cells have two different subsets of CD8 T cells expressing high (IL-7Rhigh) and low (IL-7Rlow) levels of IL-7R (shaded histogram). B and C, Rep- resentative histograms of IL-15R and IL-2/15R (shaded histograms) and isotype control (open histograms) staining. D and E, Mean fluorescence intensity (MFI) of IL-15R and IL-2/15R staining is compared between IL-7Rhigh and IL-7RlowCCR7 memory CD8 T cells (n 12). Values of p were obtained using Student’s two-tailed t test.

Article Snippet: As previously described (12), PBMCs were purified and stained with goat anti-human IL-7R Abs (R&D Systems) followed by staining with donkey anti-goat IgG Abs (R&D Systems) and Abs to CD8 and CCR7 (BD Pharmingen).

Techniques: Expressing, Staining, Cytometry, Control, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Dual roles of IL-15 in maintaining IL-7RalphalowCCR7- memory CD8+ T cells in humans via recovering the phosphatidylinositol 3-kinase/AKT pathway.

doi: 10.4049/jimmunol.179.10.6734

Figure Lengend Snippet: FIGURE 2. The gene expression of IL-2/15Rb, T-bet, and eomesoder- min in IL-7Rhigh and IL-7RlowCCR7 memory CD8 T cells. PBMCs were stained with anti-CD8, CCR7, CD45RA, IL-7R, IL-15R, IL-2/ 15R, or isotype Abs and sorted into IL-7Rhigh and IL-7RlowCCR7

Article Snippet: As previously described (12), PBMCs were purified and stained with goat anti-human IL-7R Abs (R&D Systems) followed by staining with donkey anti-goat IgG Abs (R&D Systems) and Abs to CD8 and CCR7 (BD Pharmingen).

Techniques: Gene Expression, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Dual roles of IL-15 in maintaining IL-7RalphalowCCR7- memory CD8+ T cells in humans via recovering the phosphatidylinositol 3-kinase/AKT pathway.

doi: 10.4049/jimmunol.179.10.6734

Figure Lengend Snippet: FIGURE 3. IL-15 restores limited TCR-me- diated proliferation of IL-7RlowCCR7 mem- ory CD8 T cells. PBMCs were stained with Abs to CD8, CCR7, and IL-7R and sorted into IL-7Rhigh and IL-7RlowCCR7 mem- ory CD8 T cells. A, IL-7Rhigh and IL- 7RlowCCR7 memory CD8 T cells were la- beled with CFSE and stimulated for 7 days with IL-15 or PBS. B, CFSE-labeled cells were incu- bated for 7 days in a 96-well flat-bottom tissue culture plate coated with anti-CD3 Abs (10 g/ ml) in the presence or absence of anti-CD28 Abs (10 g/ml) and/or IL-15 (5 ng/ml). Results are representative data from five independent exper- iments. Numbers on histograms indicate the fre- quency of proliferating cells.

Article Snippet: As previously described (12), PBMCs were purified and stained with goat anti-human IL-7R Abs (R&D Systems) followed by staining with donkey anti-goat IgG Abs (R&D Systems) and Abs to CD8 and CCR7 (BD Pharmingen).

Techniques: Staining, Labeling